RNA干扰Survivin基因表达对瘢痕疙瘩中成纤维细胞增殖与凋亡的影响  

Influence of expression of RNA interference mediated Survivin gene on proliferation and apoptosis of fibroblasts derived from keloid

作　　者：于冬梅[1] 尚勇[1] 王永俭 吕远东[1] 郝立君[1] 

机构地区：[1]哈尔滨医科大学附属第一医院整形美容中心,黑龙江哈尔滨150001 [2]大庆市龙南医院整形科

出　　处：《中国美容整形外科杂志》2009年第10期603-607,共5页Chinese Journal of Aesthetic and Plastic Surgery

摘　　要：目的探讨RNA干扰Survivin基因表达对瘢痕疙瘩成纤维细胞增殖与凋亡的影响，为瘢痕疙瘩的基因治疗提供实验依据。方法构建靶向Survivin的siRNA表达质粒，利用Lipofectamine^TM2000转染体外培养瘢痕疙瘩成纤维细胞；采用RT—PCR、Westernblot技术检测成纤维细胞中Survivin基因的被干扰情况及Caspase-3基因的表达情况；利用MTT法、流式细胞仪检测成纤维细胞的增殖与凋亡情况。结果靶向Survivin的序列特异性的siRNA对成纤维细胞中Survivin基因表达的抑制率；在mRNA水平为59．86％和82．57％，在蛋白质水平分别为48．76％和72．53％．Caspase-3基因的表达和活性显著增加（P〈0．01）。转染靶向Survivin的RNAi表达质粒对癜痕疙瘩成纤维细胞增殖的抑制较显著（P〈0．05）。Annexin V—FITC／PI双染法显示，干扰Survivin基因表达后成纤维细胞的凋亡率高于对照组（56．36％，P〈0．05）。结论所构建的靶向Survivin的RNAi质粒可有效地抑制瘢痕疙瘩成纤维细胞中Survivin基因的表达，通过Survivin基因表达的下调激活Caspas-3基因活性，可显著抑制成纤维细胞的增殖并诱导其凋亡，为瘢痕疙瘩的基因治疗开辟一新途径。Objective To investigate the effect of a sequence speeific small interfering RNA （siRNA） in suppressing Survivin expression and cell proliferation and inducing apoptosis of fibroblasts derived from keloid. Methods The plasmid expression veetor of siRNA targeted against Survivin was constructed and transfected into fibroblasts with Lipofcetaminc^TM 2000. The changes of Survivin expression were detected by fluorescent RT-PCR and Western blot methods. The effect of siRNA in suppressing the proliferation of fibroblasts was detected by MTT assay and flow cytometry was used for evaluation of apoptosis and modality. Results The sequence-specific siRNA effectively suppressed Survivin expression at both mRNA Revels with inhibition rates were 59.86% and 82.57% , at protein levels with inhibition rates were 48.76% and 72.53% , and enhanee the expression of easpase-3 （ P 〈0. 01 ）. Survivin expression suppression significantly inhibited the proliferation of fibroblasts （ P 〈 0. 05）. Much higher apoptosis rate than that of untreated group was also found by Annexin-V FITC/PI double staining （56.36% , P 〈0.05）. Conclusion The plasmid expression vector for the RNAi against Survivin constructed in the study can effectively and specifically suppress Survin expression in fibroblasts, and bloeking Survivin expression can suppress fibroblasts proliferation and induce cell apoptosis. RNAi targeted against Survivin has a potential value in gene therapy for keloid.

关 键 词：瘢痕疙瘩 RNA干扰 SURVIVIN CASPASE-3 细胞周期 细胞凋亡 

分 类 号：R619.6[医药卫生—外科学]